Lateral-flow test strips for detecting one or more analytes in a fluid sample generally include a reagent, or ligand, immobilized within a defined region of the test strip, variously referred to as a detection zone, a test zone, and/or a control zone. The ligand of choice has binding affinity for another reagent in the mobile phase of the test strip. If the mobile-phase reagent is detectably labeled, a detectable signal is generated within the region of the test strip occupied by its immobilized binding partner. Lateral-flow test devices generating visible signals are well known in the art. Examples of such devices are described in U.S. Pat. Nos. 5,985,675, issued Nov. 16, 1999, and 6,319,466, issued Nov. 20, 2001, the teachings of which are incorporated herein by this reference.
Lateral-flow test strips are widely used in the food products industry. For example, the United States Food and Drug Administration (FDA) requires that bulk milk tankers be tested for presence of unsafe levels of beta-lactam antibiotics. The sensitivity required pursuant to Appendix N of the Pasteurized Milk Ordinance, in parts per billion, is: amoxicillin 10 ppb, ampicillin 10 ppb, penicillin G 5 ppb, ceftiofur 50 ppb, cephapirin 20 ppb, and cloxacillin 10 ppb (the “safe levels”). The FDA also requires that the results of such tests be automatically (electronically) analyzed and recorded. It is desirable, therefore, to provide milk-testing personnel with a user-friendly test strip that can be analyzed with or without an optical reader. The test strip is used to detect beta-lactams, including five or six of the following beta-lactams at or below safe level: penicillin G, amoxicillin, ampicillin, ceftiofur, cloxacillin and cephapirin.
The sensitivity of a test device or method relative to a particular analyte initially relates to the binding affinity between the analyte and the receptor. If the receptor and analyte have good affinity, analyte-receptor complex will form readily. In the case of a multianalyte receptor, the affinity of certain analytes to the receptor may be different than others, and may not reflect the desired detection level for one or more of the analytes requiring detection. In those cases, it is desired to adjust the test sensitivity to the particular analyte. For example, U.S. Pat. No. 6,319,466 describes a test device for detecting analytes at reduced sensitivity levels. It may also be necessary to increase the sensitivity of the test to a particular analyte above the sensitivity of the multianalyte receptor.
It is an object of this invention to provide test strips with improved test zone development for use with or without an optical reading device, such as a spectrophotometer. When using an optical reading device, such improvement may involve providing consistent and uniform color development in well-defined regions of the test strip, such as test zones and control zones, for easy interpretation of results.
Various mechanisms to improve color development in test strips include adding substances to increase the viscosity of the spray solution, such as sugars, for example, sucrose, or proteins, such as bovine serum albumin (BSA). Another method is to pretreat the receptor application area. Still another method for improving the quality of the test zone is to add an abundance of label, for example, gold sol. In the case of gold, the amount used is limited by the necessity of rapid, consistent flow through the porous test strip. A high percentage of gold in the spray solution may clog pores and inhibit flow.
The above-described limitation on the use of gold is more pronounced when a second test zone is employed, for example, a second test zone for cloxacillin. In the case of a single test zone, if conjugate attachment is inefficient, or test zone development is otherwise inadequate, it may be possible to increase the concentration of label, for example gold sol, to overcome the problem. Adding a second test zone to a test strip presents special challenges when trying to optimize signal development for each zone. If there is a second test zone, which would require a second gold sol label, the technique of adding additional gold label to improve line quality is limited.